Method and kit for diagnosing or controlling the treatment of breast cancer

ABSTRACT

The present invention pertains to a procedure and a kit for the diagnosis or monitoring of breast cancer in humans. This procedure is based on the feature that one recognizes the presence or absence in a human blood sample of at least two different mRNAs that code for various members of the tumor marker proteins EGF-R, CEA, stanniocalcin, CK20, MAGE-3, GA733.2, MUC1, Her-2/neu, claudin-7, and/or PDGF-β, and a conclusion is drawn from this in regard to the presence of mammary carcinoma cells in the blood sample, and thus in regard to possible metastasis.

The present invention pertains to a procedure and a kit for thediagnosis or monitoring of breast cancer in humans.

The ability to detect a relapsing malignant tumor in a timely manner viathe occurrence of metastasizing tumor cells in the blood is relevant incancer after-care. So-called “tumor markers” are determinedquantitatively at the protein level (immunologically or enzymatically)in the blood, or in other bodily fluids, in cancer patients when usingcurrent test methods.

These detection procedures are suitable only to a limited extent fortumor diagnosis, or monitoring/after-care, since increased tumor markervalues can also be produced by nontumor diseases (e.g. inflammation ofthe gastrointestinal tract, cirrhosis of the liver, viral infections),heavy smoking, or as a result of pregnancy.

Breast cancer is the most frequent diagnosis when a tumor disease isfound in women (26.4% of all new diseases). Despite massive efforts thatare being made in regard to early detection, treatment and after-care,this disease still ranks first in cancer-related deaths in women. Thenumber of cases of this disease in the western industrialized countrieshas been increasing further over past years despite the intensifiedefforts in regard to early detection. The high rate of metastasisfollowing the initial treatment is problematical, leading to the deathof the patient after only 1-3 years in the majority of cases. The mainreason for this is the spread of tumor cells in the early stages oftumor development. Thus, in addition to the initial recognition of amammary carcinoma, the earliest possible detection of metastasizingcells is of especially decisive importance for a successful treatment.Likewise, a definitive negative detection can be helpful in clinicalstage I when one must decide whether the patient is to be subjected tothe stress of chemotherapy or an operation.

The currently used diagnostic methods are inexact when one is dealingwith the evaluation of the malignant potency of residual tumors afterchemotherapy has been carried out in the metastasizing stages. Someclinical studies indicate the prognostic importance of disseminatedtumor cells. However, numerous methodological aspects are critical andhave not been adequately standardized so far. Thus, detection methodsfor occult-or residual-metastasis have to be found that permit timelyclassification into the various primarily curative therapeutic options.

The effort to improve the chances of healing is currently accompanied,on the one hand, by the search for and the use of new tumor markers and,on the other hand, by increases in sensitivity in the methods that areused.

The problem of the present invention is to make available a procedureand a kit with which the diagnosis or monitoring of breast cancer ispossible in a simple, safe and repeatable manner.

This problem is solved by the procedure in accordance with claim 1 andwith the kit in accordance with claim 30 and the microarray inaccordance with claim 50. Advantageous further developments of theprocedure, of the kit, and of the microarray are given in the pertinentdependent claims.

In accordance with the invention, the presence or absence of mRNA fromthe tumor marker proteins EGF-R, CEA, CK20, MAGE-3, GA733.2,MUC-1≅GA15.3, Her-2/neu, claudin-7, PDGF-β and/or stanniocalcin arerecognized in a human blood sample by means of the kit in accordancewith the invention or by means of the procedure in accordance with theinvention.

Since the RNAs of the markers that have been described are not normallypresent in expressed form in the blood of healthy persons, a directcorrelation is found between a positive RT-PCR result detection forthese tumor markers and circulating tumor cells in the blood that canlead to metastasis.

Since individual markers can be expressed differently in atherapy-dependent manner, and breast cancer exhibits pronouncedheterogeneity in the expression pattern of the breast cancer cells, itis expedient to examine a combination of tumor markers in order torecognize all the tumor cells that are circulating in the blood. As aresult of this, tumor cells can also be recognized when the expressionof a particular marker is relatively slight in a patient or in a stageof the disease, which could otherwise lead to a supposedly negativeresult. However, the use of additional markers usually encounters limitsif mononuclear blood cells exhibit background expression (“illegitimatetranscription”) that impedes exact analysis.

Thus the following combination of markers is proposed in accordance withthe invention for the recognition of breast cancer cells.

Two of the markers from the following groups

-   -   GA733.2 and MUC1;    -   Her-2/neu and claudin-7;    -   CK20, MAGE-3 and MUC 1; or    -   stanniocalcin, EGFR and CEA or    -   the following combinations    -   GA733.2, MUC1, Her-2/neu, claudin-7;    -   GA733.2, PDGF-β, Her-2/neu, claudin-7;    -   GA733.2, MUC1, CEA;    -   GA733.2, PDGF-β, claudin-7; or    -   GA733.2, MUC1 and claudin-7.

Use can be made of the primers indicated in the following table for theamplification of segments of the markers.

Markers for Mammary Carcinoma Primername Sequence 5′ → 3′ PCR-ProductTumor marker GA733.2 sense AATCGTCAATGCCAGTGTACTTCA 395 bp GA733.2antisense TAACGCGTTGTGATCTCCTTCTGA EGFR sense AGTCGGGCTCTGGAGGAAAAGAAA163 bp EGFR antisense GATCATAATTCCTCTGCACATAGG CEA senseAGAAATGACGCAAGAGCCTATGTA 231 bp CEA antisense AACTTGTGTGTGTTGCTGCGGTATMUC1 sense TCAGCTTCTACTCTGGTGCACAAC 299 bp MUC1 antisenseTGGTAGTAGTCGGTGCTGGGATCT Her-2 sense CCCAGTGTGTCAACTGCAGCCAGT 265 bpHer-2 antisense CAGATGGGCATGTAGGAGAGGTCA Claudin-7 senseGTCTTGCCGCCTTGGTAGCTTGCT 225 bp Claudin-7 antisenseTGGACTTAGGGTAAGAGCGGGGTG CK20 sense ATCTCCAAGGCCTGAATAAGGTCT 336 CK20antisense CCTCAGTTCCTTTTAATTCTTCAGT MAGE3 sense CTCCAGCCTCCCCACTACCATGAA375 bp MAGE3 antisense TTGTCACCCAGCAGGCCATCGTAG Stanniocalcin senseAACCCATGAGGCGGAGCAGAATGA 254 bp Stanniocalcin antisenseCGTTGGCGATGCATTTTAAGCTCT PDGF-β sense TCTCTCTGCTGCTACCTGCGTCTG PDGF-βantisense GTTGGCGTTGGTGCGGTCTATGAG Internal Control Actin SenseCTGGAGAAGAGCTACGAGCTGCCT 111 bp Actin antisense ACAGGACTCCATGCCCAGGAAGGA

The designations of the markers are explained in the following section:Alternative Gene or gene product Gene designation Humancarcinoma-associated antigen GA733-2 GA733.2 GA733-2 gene Humanepidermal growth factor recep- EGFR EGFR tor (EGFR) gene Humancarcinoembryonic antigen CEA CEA (CEA) gene Homo sapiens mucin 1 (MUC1)MUC1 CA15-3 Homo sapiens C-erb B2/neu protein HER-2/neu HER-2 (ERBB2)gene Homo sapiens claudin 7 (CLDN7), claudin7 Claudin-7 mRNA (CLDN7)Homo sapiens gene for cytokeratin 20 CK20 CK20 Human MAGE-3 antigen(MAGE-3) MAGE-3 MAGE-3 gene Homo sapiens stanniocalcin 1 (STC1)Stanniocalcin Stanniocalcin gene (STC1) Platelet derived growth factor-βPDGF-β PDGF-β

In the case of using RT-PCR systems for the detection of tumor cells,specificity is a critical point because of the very high amplificationrate. The least contamination, e.g. via extraneous RNA or illegitimatetranscription, can in this way falsify the result.

An increase in specificity as a result of concentrating the tumor cellsrelative to the blood cells and, at the same time, an increase insensitivity in the detection of tumor cells can be achieved (detectionrate of 1 tumor cell per 10⁷ mononuclear blood cells) by usingimmunocytochemistry with monoclonal antibodies that act against tumorcell antigens. In this way the tumor cells are separated by means ofspecific antibodies or an antibody mixture of mononuclear blood cells.Separation can take place by means of magnetic particles (Dynal) towhich the antibodies are bound. This is described in greater detail inthe following segment.

Eukaryotic cells carry a plurality of different molecules on their cellsurface. The combination of expressed surface molecules differsdepending on the origin and the function of the individual cell, so thatpatterns are produced that are cell-type specific. Antibodies areutilized in order to recognize these cell-type specific patterns.Antibodies bind with high specificity to their antigen, namely toselected surface molecules in this case. This property is utilized inorder to recognize cells and to differentiate them from one another bymeans of specific antibody binding on the basis of their cell-typespecific patterns.

The expression of special surface proteins differentiates tumor cellsfrom nontransformed cells of this cell type. Since, in the case of tumorcells, this special pattern of surface antigens also differs from thepatterns that are typical of blood cells, tumor cells can bedifferentiated in the blood. In order to identify tumor cells,antibodies that specifically recognize these special surface proteinsare utilized as tools. This specific antibody binding becomes usable forvarious analyses and separation methods.

In addition to recognizing cells via their surface epitopes, it is alsopossible to separate recognized cells from nonrecognized ones because ofthe intensive binding of immunoglobulins that are specially selected forthis purpose.

1. Separation Principle Based on the Liquid Phase; e.g. Continuous FlowCytometry:

Antibodies are coupled to fluorescent dyes for the purpose of continuousflow cytometry analysis. Isolated cells are individually led past alight source (laser) in a constant stream of liquid. The fluorescentdyes bound to the antibodies are excited during illumination of thecells, and they irradiate light of a particular wavelength. Theirradiated light is detected, and the measured signal is stored indigital form. The light signal can be assigned to individual cells. Theantibody-labeled cell is recognized in this way, and can now beseparated from other cells. The cells are isolated in extremely smalldrops for separation purposes. After recognizing the antibody-labeledcell, the drops in question are deflected into a collection container.

2. Separation Principle Based on the Solid Phase; e.g. MagneticSeparation:

Antibodies are coupled to pseudomagnetic particles for the purpose ofmagnetic separation. After introducing the pseudomagnetic particles to amagnetic field, the particles migrate in this magnetic field. Duringmovement in this magnetic field, the cells to which the coupledantibodies are bound are carried along, and separated from other cells.

Thus, in order to recognize tumor cells by means of magnetic particles,antibodies are covalently coupled to pseudomagnetic particles thatpossess a defined number of chemically activated sites on their surface.The separation specificity is determined by the specificity of theantibodies. A blood sample that contains tumor cells is mixed withantibody-coupled magnetic particles; the particles and blood then moverelative to one another. Those (tumor) cells which are recognized by theantibodies (that are bound to the solid phase) and which are firmlybound to them follow the movement of the particles. As a result, it ispossible to withdraw, from the blood, the particles with the cells thatare bound to them (e.g. toward the wall of the separation vessel) uponapplying a magnetic field. The blood that has been tumor-cell depletedin this way can be exchanged for other solutions, whereby the cells thathave been separated via magnetic particles remain behind, and areavailable for additional applications until the point in time ofswitching off/removing the magnetic field.

Use can advantageously be made of specific antibody mixtures in order torecognize the tumor cells, whereby these mixtures have either beenoptimized with respect to tumor cells in a general manner, or they havebeen specifically optimized with respect to breast cancer cells as well.For example, a combination of MOC-31 antibodies (Novocastra) andBer-EP-4 antibodies (DAKO) is suitable for recognizing tumor cells inthe blood.

Recognition that is specially directed toward breast cancer cells can beachieved via an additionally optimized antibody mixture in accordancewith the table below. This is based on the selective expression ofcertain surface proteins, whereby breast cancer cells are differentiatedfrom other cancer cells. Antigen Clone Concentration Epith. Membr.Antigen 131-1174 (Hiss) 0.625 μg/10⁶ cells Epith. Membr. Antigen E29(DAKO) 0.25 μg/10⁶ cells Epith. Membr. Antigen GP1.4 (Novocastra) 18.75μg/10⁶ cells MUC-1 HMPV.2 (Pharmingen) 1.25 μg/10⁶ cells

In comparison to the antibodies when used separately in each case, suchantibody mixtures show increased sensitivity in terms of cellrecognition and cell separation, independent of the method used.

Some examples, in accordance with the invention, of detection proceduresfor breast cancer cells in blood samples will be described in thefollowing segment.

The following aspects are shown:

FIG. 1 shows the detection of PCR products via electrophoresis;

FIGS. 2A-C show tumor marker detection by means of a Light Cycler;

FIG. 3 shows the detection of cell separation by means ofantibody-labeled magnetic particles;

FIGS. 4-8 show additional examples of the detection of mammary carcinomacells by means of several tumor markers.

In the first example, the RNA from 1 mL of EDTA/whole blood wasprocessed using the QIAamp RNA Blood Mini Kit (Qiagen, Hilden).Contamination by genomic DNA was avoided via additional DNA digestion inthe column using an RNA-free DNase Set (Qiagen, Hilden).

The processing of the RNA from 1 mL of EDTA/whole blood was verifiedphotometrically via the 260:280 nm ratios. For the purposes of qualityand quantity determinations in this connection, 1 μL of the mixture canbe analyzed via electrophoretic separation on an RNA 6000 chip using theAgilent Bioanalyzer 2100.

The isolated RNA was denatured in an appropriate volume together witholigo(dT) 15 primers (Promega, Mannheim) for 5 min at 65° C., and thenincubated directly on ice. cDNA synthesis took place by means of theSensiscript™ Reverse Transcriptase Kit (Qiagen, Hilden) in 20 μL ofreaction mixture in accordance with Table 1 for 1 h at 37° C. withsubsequent reverse transcriptase inactivation for 5 min at 95° C. thatwas then followed by cooling on ice. TASBLE 1 cDNA synthesis componentsComponents Volume Final concentration RNA x μl 5 ng/μl 10 x RT buffer 2μl 1x dNTP mixture (5 mM in each case) 2 μl 0.5 mM in each case Oligo(dT)-Primer (10 μM) 2 μl 1 μM Rnase-Inhibitor 1 μl 0.5 Units/μl Reversetransciptase 1 μl 4 U RNase-free water ad 20 μl

Using the cDNA that was produced in this way, a multiplex PCR wascarried out for each of the selected tumor markers stanniocalcin, EGF-R,and CEA and also for β-actin as an internal control. The PCR mixture isillustrated in Table 2 that follows. TABLE 2 PCR mixture ComponentsVolume Final concentration CDNA 6 μl 10 x PCR buffer* 5 μl 1x dNTPmixture 1 μl 200 μM in each case Primer (See Table 3) DMO addition***1.0 μl Taq-DNA 0.5 μl 2.5 U Polymerase** H₂O ad 50 μl(*contains 15 mM MgCl₂;**HotStarTaq ™ DNA polymerase; Qiagen, Hilden***DMSO azddition in the case of stanniocalcin)

A primer pair, which is seen in Table 3 below, was used for each tumormarker in this regard. TABLE 3 List of PCR primers PCR- Primername 5′ →3′ sequence product Tumor marker Stanniocalcin AACCCATGAGGCGGAGCAGAATGA254 bp sense Stanniocalcin CGTTGGCGATGCATTTTAAGCTCT antisense EGF-Rsense AGTCGGGCTCTGGAGGAAAAGAAA 163 bp EGF-R antisenseGATCATAATTCCTCTGCACATAGG CEA sense AGAAATGACGCAAGAGCCTATGTA 231 bp CEAantisense AACTTGTGTGTGTTGCTGCGGTAT Internal control β-actin senseCTGGAGAAGAGCTACGAGCTGCCT β-actin antisense ACAGGACTCCATGCCCAGGAAGGA

The primer combinations and quantities that were used for the individualtumor marker detections are listed in Table 4 that follows. TABLE 4 Listof primer quantities and primer combinations Marker Primer StanniocalcinEGF-R CEA Stanniocalcin 25 pmol sense Stanniocalcin 25 pmol antisenseEGF-R sense 25 pmol EGF-R antisense 25 pmol CEA sense 25 pmol CEAantisense 25 pmol β-actin sense  1 pmol  1 pmol  1 pmol β-actinantisense  1 pmol  1 pmol  1 pmol

The PCR was carried out using the conditions indicated in Table 5together with the marker-specific melting temperatures and numbers ofcycles indicated in Table 6. TABLE 5 PCR conditions Vorabdenaturierung95° C. 15 min ZYklus 1. Denaturierung 94° C.  1 min 2. Annealing  x° C. 1 min (s. Table 6) 3. Extension 72° C.  1 min Finale 72° C. 10 minExtension 4° C. Pause

TABLE 6 Marker-specific annealing temperature and number of cyclesMarker Annealing Stanniocalcin EGF-R CEA Temperature 58° C. 64° C. 60°C. Number of cycles 35 35 40

1 μL of the PCR product produced in this way was separated in an AgilentBioanalyzer 2100 on a DNA chip (500), and the result of the separationwas documented electronically. The results are shown in FIG. 1. In thisdiagram, lane 1 shows a 100 kb ladder, and lanes 2-13 show the resultsfor, the corresponding samples. As can be seen, lane 5 shows a PCRproduct for the tumor marker stanniocalcin; lane 9 shows a PCR productfor the tumor marker EGF-R, and lane 13 shows a PCR product for thetumor marker CEA, whereas all samples with a biological material inaccordance with lanes 4, 5, 8, 9, 12 and 13 contain PCR products for theinternal control β-actin.

Lanes 2, 3, 6, 7, 10, 11 do not contain any biological material, so thatno corresponding PCR products arise there. In FIG. 1, the so-called cDNAcontrol is a mixture totally without RNA; the so-called PCR control is amixture without cDNA, and the negative control is a mixture containingRNA from a healthy control person. In FIG. 1, CEA stands forcarcinoembryonic antigen; STC stands for stanniocalcin, and EGF-R standsfor epidermal growth factor receptor.

FIG. 2 shows an alternative analysis by means of fluorescence-based realtime PCR using intercalating fluorescent dyes.

As an alternative to block PCR, this tumor marker detection procedurecan also be done by means of a Light Cycler (Roche, Basel).

Reverse transcription of the mRNA was done as described above. The PCRwas then carried out with the Light Cycler DNA Master Sybr Green I′ Kit(Roche, Basel) in accordance with data from the manufacturer underconditions that had been optimized for each tumor marker. Theoligonucleotides that are indicated in Table 3 were used as primers inthis case. Table 7 and Table 8, respectively, show the mixture for thePCR and the PCR conditions using the Light Cycler. TABLE 7 PCR mixture:Light Cycler Tumor marker Components Stanniocalcin EGF-R CEA CDNA 3.0 μl3.0 μl 3.0 μl MgCl₂ 3.0 mM 3.5 mM 3.5 mM Primer 0.5 μM 0.5 μM 0.5 μMLight Cycler- 2 μl 2 μl 2 μl DNA Master Sybr Green DMSO 1 μl — — H₂O ad20 μl azd 20 μl ad 20 μl

TABLE 8 PCR conditions: Light Cycler Stanniocalcin EGFR CEA Denaturation95° C., 30 Sec 95° C., 95° C., 20° C./Sec 30 Sec 30 Sec 20° C./Sec 20°C./Sec Amplification 95° C., 5 Sec, 95° C., 95° C., 20° C./Sec 5 Sec, 5Sec, 20° C./Sec 20° C./Sec 67° C., 10 Sec, 60° C., 60° C., 20° C./Sec 10Sec, 20° C./ 10 Sec, 20° C./Sec 73° C., 15 Sec, 73° C., 72° C., 5°C./Sec 15 Sec, 12 Sec, 5° C./Sec 5° C./Sec Melting Curve 95° C., 0 Sec,95° C., 95° C., 20° C./Sec 0 Sec, 0 Sec, 20° C./Sec 20° C./Sec 70° C.,20 Sec, 65° C., 65° C., 20° C./Sec 20 Sec, 15 Sec, 20° C./Sec 20° C./Sec95° C., 0 Sec, 95° C., 95° C., 0, 1° C./Sec 0 Sec, 0 Sec, 0, 1° C./Sec0, 1° C./Sec Cooling 30° C., 30 Sec 30° C., 30° C., 20° C./Sec 30 Sec 30Sec 20° C./Sec 20° C./Sec

The result of this PCR and the evaluation using Light Cycler technologyare illustrated in FIGS. 2A through 2C. The control curve is designated2 in all the FIGS. 2A through 2C, whereas the curve that was recordedfor the sample is designated 1.

In this analysis, the melting curve for the PCR products is analyzed bymeans of the Sybr Green I detection method. The pertinent graph in FIGS.2A through 2C is the fluorescence that was measured as a function of thetemperature. The fluorescence peaks that occur in the control mixturesare attributable to primer dimers.

FIG. 2A represents the melting curve analysis of the stanniocalcin PCRproduct. The melting point of the main product is 89.2° C., and themelting point of the secondary product is 85.3° C. Such fluorescencepeaks cannot be seen in the case of the control sample.

FIG. 2B shows the melting curve analysis of the EGFR PCR product with amelting point of 84.6° C.

FIG. 2C shows the melting curve analysis of the CEA PCR product with amelting point of 89.06° C.

As an alternative to the methods that are illustrated here, use can, ofcourse, be made of conventional methods of analysis as well, such asagarose gel electrophoresis in which, for example, 25 μL of the PCRproduct synthesized above are separated over a 2.5% agarose gel, and theDNA bands are then stained with ethidium bromide and rendered visible.Documentation can be carried out with the help of e.g. the DUO StoreSystem from Intas.

In addition, fragment analysis by means of the ABI Prism 310 GeneticAnalyzer (Applied Biosystems, Weiterstadt) can also be used for theevaluation. In order to do this, a PCR is carried out withfluorescence-labeled primers and then, for example, 1 μL in each case ofeach PCR product is used at a dilution of 1:50.

Detection by means of sequence-specific fluorescence-labeledhybridization samples is possible as an additional detection procedure,whereby these samples allow the evolution of products to be monitoredafter each PCR cycle. A conclusion can then be drawn on the basis ofspecial standards in regard to the quantity of starting RNA.

The enrichment of the cell fraction which is used for this purpose andwhich arises from the blood sample used is central for the quality ofthe RNA, which is isolated as the basis of the detection procedure, andthe cDNA that is synthesized therefrom. Four different methods areavailable for this as follows.

a) Enrichment by Means of Repeated Centrifugation Following ErythrocyteAnalysis:

1 mL of EDTA/blood is lysed for 20 min on ice following the addition of5 volumes of erythrocyte lysis buffer (“QIAmp Blood Kit,” Qiagen;Hilden). The plasmallysate is removed from the pelletized cells andresuspended, and then renewed centrifugation takes place for 20 min at3000×g. After removing the supernatant liquor, the pelletized leukocytefraction is available for RNA preparation.

b) Enrichment by Means of Density Gradient Centrifugation:

Cells of different mean volume-based density can be separated from oneanother via a density gradient that is produced by means ofcentrifugation. Mononuclear blood cells are separated by means of aFicoll-Hypaque gradient (Pharmacia, Uppsala, Sweden), and then washedtwice with PBS/1% FCS.

c) Enrichment of Tumor Cells by Means of FACS Continuous Flow Cytometry:

The mononuclear cells from the fraction enriched under b) are incubatedwith fluorescence-labeled mononuclear antibodies that act againsttumor-specific surface proteins. The labeled cells are washed twice withPBS, and then 10⁷ cells are resuspended in 1 mL of PBS. A FACS VantageSE continuous flow cytometer (Becton Dickinson) is used in order toisolate the tumor cells. Data recording, instrument control, and dataevaluation are done via the CellQuest program. The sorted cells aretransferred to a 1.5-mL reaction vessel (filled with 1 mL of PBS). TheRNA can then be isolated as described above.

As an alternative, the isolated fraction of mononuclear blood cells,which were isolated in accordance with one of the above procedures, waslysed in trizole reagent (Gibco BRL, New York, USA), and homogenized bymeans of a pipette. Following chloroform extraction, the RNA-containingaqueous phase is precipitated in isopropanol at −80° C. After washingtwice in 80% ethanol, the pellet is dried in air, and then resuspendedin RNase-free water.

Reverse transcription and mRNA detection as described above then followon from this isolation of the RNA.

d) Enrichment of Tumor Cells by Means of Immunomagnetic Separation:

The expression of special surface proteins differentiates tumor cellsfrom nontransformed cells of this cell type. Since, in the case of tumorcells, this special pattern of the surface antigens is also differentfrom the patterns that are typical of blood cells, one can differentiatebetween tumor cells in the blood. In order to identify tumor cells,antibodies which specifically recognize these special surface proteinsare utilized as tools. Specific antibody binding is made usable for theprocedure in accordance with the invention. Antibodies are covalentlycoupled to pseudomagnetic particles that possess a defined number ofchemically activated sites on their surface. The separation specificityis determined by the specificity of the antibodies. A blood sample thatcontains tumor cells is mixed with antibody-coupled magnetic particles;two different mixtures of antibodies are used as antibodies in thevarious examples; the particles and blood then move relative to oneanother, e.g. by means of “over-end rotators” in samples that arelocated in a closed container or by means of alternating magneticfields. Those (tumor) cells which are recognized by the antibodies (thatare bound to the solid phase) and which are firmly bound to them followthe movement of the particles. As a result, it is possible to withdraw,from the blood, the particles with the cells that are bound to them(e.g. toward the wall of the separation vessel) upon applying a magneticfield. The blood that has been tumor cell depleted in this way can beexchanged for other solutions, whereby the cells that have beenseparated via magnetic particles remain behind, and are available foradditional applications until the point in time of switchingoff/removing the magnetic field. TABLE 9 Antibody mixture 1 AntigenClone Concentration Epith. Rel. Antigen MOC-31 (Fa. Novocastra) 1.25μL/10⁶ cells Epithelial antigen Ber-EP 4 (Fa. DAKO) 0.924 μg/10⁶ cells

However, tumor cells were recognized with high specificity quitegenerally by means of the antibody mixture in Table 9. This is based onthe selective expression of certain surface proteins that differentiatecancer cells from other cells.

In comparison to the separately used antibodies, an increasedsensitivity during cell separation was demonstrated quite invariably,and independently of the method used, as a result of the use of theantibody mixture. This is shown in FIG. 3, whereby use was made insubdiagram A of magnetic particles that were coated with the antibodyBER-EP4, and whereby use was made in subdiagram B of magnetic particlesthat were coated with the antibody MOC-31, and whereby use was made insubdiagram C of a mixture comprising particles that were each separatelycoated with an antibody.

A total of four measurements, in which, respectively, 1, 10, 100, or1000 carcinoma cells in 10 mL of blood were inoculated, were carried outfor each of the antibodies or antibody mixtures. Lanes 1a through 4a, 1bthrough 4b, and 1c through 4c then show the detection of RNA followingRNA preparation and RT-PCR with tumor marker-specific primers, asdescribed above, for samples with a volume of 1 μL in each case. FIG. 3was obtained by means of electrophoretic separation in an Agilent™Bioanalyzer 2100 in accordance with data from the manufacturer.

When using magnetic particles labeled with merely one antibody as inFIGS. 3A and 3B, positive detection was possible only with a quantityamounting to 1000 cells. When using an antibody mixture as in FIG. 3C,detection was achieved with only 100 cells, i.e. an increase insensitivity by a factor of 10.

In this example, experimental results have been shown that do notrepresent the maximum possible sensitivity but, by way of example, theydemonstrate the increase in sensitivity that is achievable with theprocedure in accordance with the invention.

FIG. 4 shows the detection of breast cancer cells via the simultaneousrecognition of tumor cell markers GA733.2, MUC1, Her-2 and claudin-7. Inthis case, breast tumor cells in a sample were inoculated, wherebydifferent cell lines, namely cell line 1 and cell line 2, wereintroduced. Enrichment of the tumor cells by means of antibody-coupledmagnetic particles took place prior to the determination of the markers,whereby use was made of the antibodies BerEp4, HMTV.2 and GP1.4. FIG. 4then shows that, for both cell lines, sure recognition can bedemonstrated down to two cells per 5 mL by means of such a combinationof the tumor markers that were to be recognized. A nonspecific reactiondid not take place in this regard. In the following segment, the PCRconditions are illustrated for the different polymerase chain reactionsshown in FIG. 4 through FIG. 8 in order to detect tumor markers frombreast cancer cells. Standard, FIG. 4 Marker Primer Concentration Actinsense 0.1 μM antisense 0.1 μM Claudin-7 sense 0.3 μM anrtisense 0.3 μMHer-2 sense 0.3 μM antisense 0.3 μM MUC1 sense 0.4 μM antisense 0.4 μMGA733.2 sense   1 μM antisense   1 μMCycles: 35

Multiplex 1, FIG. 5A Marker Primer Concentration Actin sense 0,1 μMantisense 0,1 μM Claudin-7 sense 0,3 μM antisense 0,3 μM PDGF-β sense1,2 μM antisense 1,2 μM Her-2 sense 0,3 μM antisense 0,3 μM GA733.2sense   1 μM antisense   1 μMCycles: 35

Multiplex 2, FIG. 5B Marker Primer Concentration Actin sense 0,1 μMantisense 0,1 μM GA733.2 sense   1 μM antisense   1 μM MUC1 sense 0,4 μMantisense 0,4 μM CEA sense   2 μM antisense   2 μMCycles: 35

Multiplex 3, FIG. 6 Marker Primer Concentration Actin sense 0,1 μMantisense 0,1 μM GA733.2 sense   1 μM antisense   1 μM PDGF-β sense 1,2μM antisense 1,2 μM Claudin-7 sense 0,3 μM antisense 0,3 μMCycles: 35

Multiplex 4, FIG. 7 Marker Primer Concentration Actin Sense 0,1 μMAntisense 0,1 μM Claudin-7 Sense 0,3 μM Antisense 0,3 μM MUC1 Sense 0,4μM Antisense 0,4 μM GA733.2 Sense   1 μM Antisense   1 μMCycles: 35

Multiplex 5, FIG. 8A Marker Primer Concentration Actin sense 0,05 μMantisense 0,05 μM GA733.2 sense 0,05 μM antisense 0,05 μM PDGF-β sense005 μM antisense 0,05 μM CEA sense  0,7 μM antisense  0,7 μMCycles: 40

FIG. 5, likewise, shows the detection of breast tumor cells, whereby thecombinations comprising the markers GA733.2, PDGF-β, Her-2 and claudin-7(FIG. 5A) or GA733.2, MUCL and CEA were recognized. Highly sensitiverecognition takes place once again down to two cells per 5 mL sample,whereas no nonspecific detection reactions occurred in a control bloodsample.

FIG. 6 illustrates the use of the marker combination GA733.2, PDGF-β andclaudin-7 (FIG. 6A), or GA733.2, PDGF-β and claudin-7 (FIG. 6B) for afirst cell line (FIG. 6A), or a second cell line (FIG. 6B). Highlyspecific detection again takes place without nonspecific reactions,whereby cell line 2 can be detected better than cell line 1 in thiscase. FIG. 7 again illustrates a detection procedure by means of thecombination of the markers GA733.2, MUC1 and claudin-7. Highly specificdetection takes place for cell line 2 down to two cells per 5 mL samplefor each individual marker and, in particular, for the combination ofthe markers, without nonspecific detection reactions.

FIG. 8 shows the detection procedure by means of the marker combinationGA733.2, PDGF-β and CEA for cell line 2. Highly specific detection againtakes place down to two cells per 5 mL sample without nonspecificdetection reactions.

The diagnosis kit in accordance with the invention and the procedure inaccordance with the invention also make it possible to subsequently usethe sorted and separated cells further as desired. For example, thesecan be inserted into a suitable cell culture medium where they can becultivated in situ.

Since the cells are intact following separation, the properties of thecell membrane and of the cell nucleus are also conserved. This opens upthe possibility of microscopically investigating the expression ofadditional surface markers, and of carrying out chromosome analyses aswell. The sorted cells are applied to microscope slides for thispurpose. The detection of additional surface markers can take placecytochemically or via fluorescence microscopy. Likewise, geneticanalyses can be carried out such as, for example, chromosome analyses bymeans of FISH (fluorescence in situ hybridization), or via karyogramcompilation.

1. Procedure for the diagnosis or monitoring of breast cancer in humans,characterized in that the presence or absence of at least threedifferent mRNAs is recognized in a human blood sample, whereby thesecode for various members of the tumor marker proteins GA733.2, MUC1 andHer-2/neu and, if at least one of the mRNAs is present, conclusions aredrawn in regard to the presence of mammary carcinoma cells in the sampleof blood, and hence in regard to possible metastasis.
 2. Procedure inaccordance with claim 1, characterized in that the presence or absenceof additional mRNAs is recognized that code for various members of thetumor marker proteins claudin-7, CK20, MAGE-3, stanniocalcin, EGF-Rand/or CEA.
 3. Procedure in accordance with the preceding claim,characterized in that the mRNA associated with the genes GA733.2, MUC1,Her-2/neu and claudin-7 is recognized.
 4. Procedure in accordance withone of the preceding claims, characterized in that tumor cells from theblood sample are separated or concentrated, and the detection procedureis done using these tumor cells.
 5. Procedure in accordance with thepreceding claim, characterized in that the mammary carcinoma cells areseparated or concentrated by means of antibodies, which are generallyspecific for tumor cells, and/or by means of antibodies specific formammary carcinoma cells or by means of mixtures of such antibodies. 6.Procedure in accordance with the preceding claim, characterized in thatthe antibodies or antibody derivatives used for separating mammary tumorcells have binding sites that bind to the epitopes of an epithelialantigen of an epithelial membrane antigen and/or of the antigen MUC1. 7.Procedure in accordance with the preceding claim, characterized in thatuse is made of MOC-31 and/or Ber-EP4, or a mixture of these, as theantibody.
 8. Device in accordance with one of the preceding claims,characterized in that use is made of 131-11741, E29, GP1.4 and/orHMPV.2, or a mixture of all of these, as the antibody.
 9. Procedure inaccordance with one of the two preceding claims, characterized in thatthe mammary carcinoma cells are separated or concentrated by means ofantibodies bound to magnetic particles.
 10. Procedure in accordance withclaim 4, characterized in that the mammary carcinoma cells are separatedor concentrated by means of fluorescence-activated continuous flowcytometry, density gradient centrifugation, and/or centrifugationfollowing erythrocyte lysis.
 11. Procedure in accordance with claim 10,characterized in that leukocytes in the blood sample are pelletized bycentrifugation.
 12. Procedure in accordance with claim 10, characterizedin that the RNA-containing components of the sample are concentrated vialysis of the erythrocytes that are contained in it, together withsubsequent pelletization of the nonlysed leukocytes.
 13. Procedure inaccordance with claim 10, characterized in that the RNA-containingcomponents are concentrated by at least one density gradientcentrifugation of the blood sample in order to separate and harvest themononuclear blood cells that are contained in it.
 14. Procedure inaccordance with one of claims 10 through 13, characterized in that theharvested mononuclear blood cells are labeled with fluorescence-labeledantibodies and are separated and harvested by means offluorescence-activated cell sorting (FACS) of the sample.
 15. Procedurein accordance with one of claims 10 through 14, characterized in thatthe mononuclear cells from the harvested fraction are lysed, and themRNA is separated.
 16. Procedure in accordance with claim 1,characterized in that the RNA (total RNA or mRNA) is isolated directlyand in a conventional manner from the whole blood sample.
 17. Procedurein accordance with the preceding claim, characterized in that DNAdigestion is carried out subsequently to isolation of the RNA. 18.Procedure in accordance with one of the preceding claims, characterizedin that the harvested mRNA is reverse transcribed into cDNA, and thepresence or absence of the cDNA that is assigned to the tumor markerprotein is recognized.
 19. Procedure in accordance with the precedingclaim, characterized in that at least one predetermined segment of thecDNA is replicated by means of a polymerase chain reaction (“PCR”). 20.Procedure in accordance with the preceding claim, characterized in thatone or more oligonucleotide pairs which exhibit the following sequencesare used for replicating the cDNA: AATCGTCAATGCCAGTGTACTTCA andTAACGCGTTGTGATCTCCTTCTGA and/or AGTCGGGCTCTGGAGGAAAAGAAA andGATCATAATTCCTCTGCACATAGG and/or AGAAATGACGCAAGAGCCTATGTA andAACTTGTGTGTGTTGCTGCGGTAT and/or TCAGCTTCTACTCTGGTGCACAAC andTGGTAGTAGTCGGTGCTGGGATCT and/or CCCAGTGTGTCAACTGCAGCCAGT andCAGATGGGCATGTAGGAGAGGTCA and/or GTCTTGCCGCCTTGGTAGCTTGCT andTGGACTTAGGGTAAGAGCGGGGTG and/or ATCTCCAAGGCCTGAATAAGGTCT andCCTCAGTTCCTTTTAATTCTTCAGT and/or CTCCAGCCTCCCCACTACCATGAA andTTGTCACCCAGCAGGCCATCGTAG and/or AACCCATGAGGCGGAGCAGAATGA andCGTTGGCGATGCATTTTAAGCTCT and/or TCTCTCTGCTGCTACCTGCGTCTG andGTTGGCGTTGGTGCGGTCTATGAG


21. Procedure in accordance with one of the preceding claims,characterized in that the mRNA of the protein β-actin is determined forinternal control purposes.
 22. Procedure in accordance with thepreceding claim, characterized in that the mRNA for β-actin is reversetranscribed into cDNA, and a segment of the cDNA is replicated by meansof a polymerase chain reaction.
 23. Procedure in accordance with thepreceding claim, characterized in that an oligonucleotide pair is usedfor replicating the cDNA of the β-actin, whereby the oligonucleotides ofthe pair exhibit the following sequences: CTG GAG AAG AGC TAC GAG CTGCCT and ACA GGA CTC CAT GCC CAG GAA GGA.


24. Procedure in accordance with one of claims 19 through 23,characterized in that the replicated cDNA segment is digested viasuitable restriction enzymes, and the presence or absence of the mRNA ofa tumor marker protein is determined by means of the cDNA fragments thatare produced.
 25. Procedure in accordance with one of claims 19 through23, characterized in that gel electrophoresis of the PCR products iscarried out in order to detect the amplified cDNA segments. 26.Procedure in accordance with one of claims 19 through 23, characterizedin that fragment analysis is carried out in order to detect theamplified cDNA segments.
 27. Procedure in accordance with one of claims19 through 23, characterized in that, during the course of thepolymerase chain reaction, the fluorescence produced by the products isrecognized and the formation of products is recognized(fluorescence-based real time PCR).
 28. Procedure in accordance with oneof claims 19 through 23, characterized in that use is made of anucleotide microarray in accordance with one of claims 48 through 50 inorder to detect the mRNA or cDNA.
 29. Procedure in accordance with thepreceding claim, characterized in that the PCR product is applied to anucleotide microarray in accordance with one of claims 48 through 50 inorder to detect the amplified cDNA.
 30. Diagnosis kit for the diagnosisor monitoring of breast cancer via at least three pairs ofoligonucleotides (reverse primers, forward primers), whereby the twooligonucleotides of each pair are suitable as primers for amplificationby means of a polymerase chain reaction of, in each case, one of the twocomplementary strands of different DNA segments that are being sought,and whereby the DNA segments that are being sought are a part of thecDNA associated with various members of the genes GA733.2, MUC-1 andHer-2/neu.
 31. Diagnosis kit in accordance with claim 30, characterizedin that it contains at least one additional pair of oligonucleotides(reverse primers, forward primers), whereby the two oligonucleotides ofeach pair are suitable as primers for amplification by means of apolymerase chain reaction of, in each case, one of the two complementarystrands of a DNA segment that is being sought, and whereby the DNAsegment that is being sought is a part of the cDNA associated with oneof the tumor marker proteins CK20, EGF-R, CEA and stanniocalcin, orCK20, MAGE-3, claudin-7, and/or PDGF-β.
 32. Diagnosis kit in accordancewith claim 30, characterized in that it contains at least four pairs ofoligonucleotides (reverse primers, forward primers), whereby the twooligonucleotides of each pair are suitable as primers for amplificationby means of a polymerase chain reaction of, in each case, one of the twocomplementary strands of different DNA segments that are being sought,and whereby the DNA segments that are being sought are, in each case, apart of the cDNA associated with the tumor marker proteins GA733.2,MUC-1, Her-2/neu and claudin-7.
 33. Diagnosis kit in accordance with oneof the two preceding claims, characterized in that it contains anadditional pair of oligonucleotides that are, in each case, suitable asprimers for the amplification of at least one segment of the twocomplementary strands of the cDNA associated with the protein β-actinfor internal control purposes.
 34. Diagnosis kit in accordance with oneof claims 30 through 33, characterized in that the two oligonucleotidesof a pair exhibit the following sequences in a pair-wise manner:AATCGTCAATGCCAGTGTACTTCA and TAACGCGTTGTGATCTCCTTCTGA and/orAGTCGGGCTCTGGAGGAAAAGAAA and GATCATAATTCCTCTGCACATAGG and/orAGAAATGACGCAAGAGCCTATGTA and AACTTGTGTGTGTTGCTGCGGTAT and/orTCAGCTTCTACTCTGGTGCACAAC and TGGTAGTAGTCGGTGCTGGGATCT and/orCCCAGTGTGTCAACTGCAGCCAGT and CAGATGGGCATGTAGGAGAGGTCA and/orGTCTTGCCGCCTTGGTAGCTTGCT and TGGACTTAGGGTAAGAGCGGGGTG and/orATCTCCAAGGCCTGAATAAGGTCT and CCTCAGTTCCTTTTAATTCTTCAGT and/orCTCCAGCCTCCCCACTACCATGAA and TTGTCACCCAGCAGGCCATCGTAG and/orAACCCATGAGGCGGAGCAGAATGA and CGTTGGCGATGCATTTTAAGCTCT and/orTCTCTCTGCTGCTACCTGCGTCTG and GTTGGCGTTGGTGCGGTCTATGAG.


35. Diagnosis kit in accordance with one of claims 30 through 34,characterized in that, in each case, at least one of the twooligonucleotides of a pair of oligonucleotides is labeled withfluorophores.
 36. Diagnosis kit in accordance with the preceding claim,characterized in that the oligonucleotides of different pairs arelabeled with different fluorophores.
 37. Diagnosis kit in accordancewith one of claims 30 through 36, characterized in that, in order toamplify the cDNA associated with β-actin, it contains a pair ofoligonucleotides with the following sequences: CTG GAG AAG AGC TAG GAGCTG GGT and ACA GGA CTC CAT GGG GAG GAA GGA.


38. Diagnosis kit in accordance with the preceding claim, characterizedin that, in each case, at least one of the two oligonucleotides of thepair is labeled with fluorophores in order to amplify the cDNAassociated with β-actin.
 39. Diagnosis kit in accordance with one ofclaims 30 through 38, characterized in that it contains the substancesthat are required for carrying out a polymerase chain reaction. 40.Diagnosis kit in accordance with one of claims 30 through 39,characterized in that it contains the following as the substances thatare required for carrying out a polymerase chain reaction: a buffersolution, magnesium chloride, deoxynucleotide triphosphates as well as aheat-stable polymerase.
 41. Diagnosis kit in accordance with thepreceding claim, characterized in that it contains a polymerase fromThermus aquaticus (Taq polymerase) as the heat-stable polymerase. 42.Diagnosis kit in accordance with one of claims 30 through 41,characterized in that, as a positive control, it contains a DNA samplewith the DNA segment that is being sought in each case.
 43. Diagnosiskit in accordance with one of claims 30 through 42, characterized inthat it contains: instructions for carrying out the polymerase chainreaction and/or instructions for carrying out a fragment analysis. 44.Diagnosis kit in accordance with one of claims 30 through 43,characterized in that it contains a schematic arrangement for evaluatingthe measurement results.
 45. Diagnosis kit in accordance with one ofclaims 30 through 44, characterized in that it contains a microarray(DNA chip), whereby the array has a number of cells (fields) which areseparated from one another, and an oligonucleotide, which hybridizeswith the DNA segment that is being sought, is arranged in at least onecell of the microarray.
 46. Diagnosis kit in accordance with thepreceding claim, characterized in that an additional oligonucleotide isarranged in at least one additional cell of the microarray, and thesequence of the nucleotide arranged in said cell differs from thesequence of the additional oligonucleotide.
 47. Diagnosis kit inaccordance with one of the two preceding claims, characterized in thatan oligonucleotide is arranged in at least two cells in each case,whereby the oligonucleotides are arranged in different cells hybridizein each case with the different DNA segments that are being sought. 48.Microarray for the diagnosis or monitoring of breast cancer, e.g. a DNAchip, with an arrangement of several cells that are separated from oneanother, characterized in that, in each case, different oligonucleotidesare arranged in at least three cells, whereby these oligonucleotideshybridize with a DNA segment that is part of the cDNA associated withthe three different tumor marker proteins GA733.2, MUC-1 and Her-2/neu.49. Microarray in accordance with claim 48, characterized in thatdifferent oligonucleotides are arranged in at least four cells in eachcase, whereby these oligonucleotides hybridize in each case with fourdifferent DNA segments that are part of the cDNA of the tumor markerproteins GA733.2, MUC-1, Her-2/neu and claudin-7.
 50. Microarray inaccordance with claim 48 or 49, characterized in that oligonucleotidesare arranged in additional cells, whereby these oligonucleotideshybridize with DNA segments that are part of the cDNA of the tumormarker proteins CK20, EGF-R, CEA, stanniocalcin, MAGE-3 and/or PDGF-β.51. Use of a diagnosis kit, a microarray and/or a procedure inaccordance with one of the preceding claims for the diagnosis ofdiseases or metastasis or for monitoring in cases of breast cancer.